Abstract: Pancreatic beta-cells are the last line of defense to preserve glucose homeostasis and preventing diabetes. Some past therapies that have enhanced their function are associated with a loss of durability due in part to injury and dedifferentiation of beta-cells. A clear understanding of the metabolic features that are tied improved function as well as those that are detrimental may help in the development of new therapies. Much of the understanding of how glucose is metabolized to generate a signal to release insulin have been obtained in a piecemeal fashion. As a consequence, there has been a surprising divergence, rather than a convergence, on the fundamentals such as metabolism-secretion-coupling as well as metabolic toxicities. New quantitative and comprehensive methods are required to reevaluate the relationship of flux through metabolic pathways in human beta-cells. The Kibbey lab has recently developed such a platform called Mass Isotopomer Multi Ordinate Spectral Analysis (MIMOSA) that can follow the stepwise transfer of mass isotope labeled substrates through glycolysis and the TCA cycle. Here a proposed expansion of this innovation to include additional metabolic flux measurements will assess normal and diabetic human beta-cells. MIMOSA will first be applied to characterize the fundamentals of normal beta-cell metabolism in response to different fuels, metabolic stimuli, and medicines. A second aim will follow up on the observation that glucokinase activators restore insulin secretion in diabetic humans but ultimately loose durability due to toxic metabolism. Here the top-down ?pushing? metabolism will be compared to ?pulling? metabolism from the bottom using small molecule enzymatic activators. Preliminary data using MIMOSA identifies an import benefit of activating anaplerotic pyruvate carboxylase metabolism in normal, glucolipotoxic, and diabetic human islets. However, top-down pushing leads to overflow of glycolytic metabolites into detrimental metabolic pathways that are relieved by pharmacologically unloading glycolysis. A third aim will translate these findings in vivo, where activation of the allosterically-regulated pyruvate kinase isoforms are anticipated to improve glucose homeostasis both by stimulating insulin secretion but also uncoupling gluconeogenesis via energy wasting futile cycles that improve insulin sensitivity. So taken together, this proposal leverages an innovative metabolic flux platform to identify the mechanistic fundamentals of how beta-cells work and how they fail and translates this information in vivo to validate a potential novel therapeutic approach to treat diabetes.